Protein kinases, the largest family of human enzymes, encompass well over 500 proteins. Kinases play critical roles in signaling pathways controlling fundamental cellular processes such as proliferation, differentiation, and death (apoptosis). Abnormal kinase activity has been implicated in a wide range of diseases, including multiple cancers and autoimmune and inflammatory diseases. The multifaceted role of kinases in key cell signaling pathways provides a significant opportunity to identify novel drugs targeting kinases and signaling pathways. Diseases mediated by receptor kinase activity include, but are not limited to, diseases characterized in part by abnormal levels of cell proliferation (i.e. tumor growth), programmed cell death (apoptosis), cell migration and invasion, and angiogenesis associated with tumor growth.
The recently demonstrated efficacy of multiple kinase inhibitors in the treatment of cancer, including the FDA approval of the kinase inhibitor GLEEVEC (imatinib mesylate), a c-Kit, PDGFR, and Ab1 kinase inhibitor, for the treatment of chronic myeloid leukemia, and the proof of clinical efficacy for AVASTIN, a VEGF modulator that inhibits angiogenesis, is testimony to the great clinical potential of kinase and other signal transduction inhibitors as therapeutics.
Kinases also play a key role in angiogenesis. Angiogenesis, the formation of new blood vessels from preexisting ones, plays a critical role in many pathological settings, including cancer, chronic inflammation, diabetic retinopathy, psoriasis, rheumatoid arthritis, and macular degeneration. Anti-angiogenic therapy represents a potentially important approach for the treatment of solid tumors and other diseases associated with dysregulated vascularization.
Angiogenesis is regulated by multiple cell-signaling pathways, including pathways controlled by cellular kinases. Blocking angiogenesis, through the modulation of cell kinases, therefore, represents an effective approach to the treatment of diseases such as cancer.
The process of angiogenesis is complex, requiring the concerted actions of multiple angiogenic mediators as well as the participation of different cell types. Key angiogenesis mediators, including, VEGF, FGF, and angiopoietin 1 and 2 (Ang1 and Ang2) that bind to their cognate receptors (VEGFRs, FGFRs and Tie1 and Tie2, respectively) expressed on endothelial cells, as well as platelet-derived growth factor (PDGF) that binds to its receptor (PDGFRs) expressed on pericytes and smooth muscle cells have been identified. Recent studies indicate that several members of the ephrin family and their receptor Eph family are novel regulators of angiogenesis.
Because tumor angiogenesis is a complex process, maximum blockage of tumor angiogenesis, leading to tumor stasis and/or eradication, is most likely to be achieved by simultaneously modulating multiple angiogenesis mediators. The VEGFR2, Tie-2, PDGFRβ, EphA2 EphB2, EphB4, and FGFR1-4 receptors, as well as other kinases, are believed to be involved in angiogenesis. Thus, modulation of these receptors, or other kinases implicated in the angiogenesis process, is desirable for treating diseases such as cancer, in which angiogenesis plays a role.
Agents capable of modulating angiogenic kinases, especially those capable of modulating each of EphB4, VEGF-R2, and Tie-2, are highly desirable for the treatment of a variety of diseases and disorders, including cancer and diseases and disorders characterized by pathological angiogenesis. Small molecule, non-peptide antagonists of angiogenic kinases are of particular value for such therapies. The present invention fulfills this need, and provides further related advantages.
Many of the cellular processes regulated by kinases are further regulated by Hsp90.
Hsp90 is a molecular chaperone, a class of proteins that regulates protein folding in cells. Hsp90 is a 90 kD protein that functions as a homodimer. Hsp90 regulates its own expression by sequestering the transcription factor, HSF1, under non-stress conditions. Upon heat shock, HSF1 is released from Hsp90 leading to transcription and increased synthesis of Hsp90, thereby controlling the cellular stress response.
Numerous contacts in the 190 C-terminal amino acids of the protein are responsible for dimerization of this protein. The 25 kD NH2-terminal of Hsp90 contains an ATP binding site, where ATP is bound and subsequently hydrolyzed. Thus Hsp90 is an ATPase, and has been classified as a member of the GHKL ATPase superfamily. It is believed that unfolded, or partially folded substrate proteins, also called Hsp90 client proteins, are stably bound to Hsp90 in its ATP bound state, and released upon ATP hydrolysis.
Hsp90 is an important cell cycle regulatory protein, implicated in the correct folding of multiple proteins in the mitogenic signal cascade. Hsp90 also plays a role in cyclin dependent progression through G1 and G2 and in centrosome function in mitosis. Hsp90 substrates include a number of steroid hormone receptors including the androgen receptor (AR), estrogen receptor, and glucocorticoid receptor.
Hsp90 has been specifically implicated in the proper folding of a number of tyrosine and threonine kinases. It also insures the correct folding and activity of numerous kinases involved in cell proliferation and differentiation, many of which also play roles in oncogenesis.
Hsp90 can also function as part of a multi-component complex interacting with many other co-chaperone proteins. While Hsp90 forms a multi-component complex to some extent in normal cells, nearly all Hsp90 present in cultured tumor cells has been shown to be part of a multi-component complex. A number of known oncogenic proteins that are Hsp90 substrate proteins, depend on the chaperone activity of the Hsp90 complex for correct folding. Thus Hsp90 functions as a supplier of oncogenic proteins in tumor cells. Hsp90 complex in tumor cells also exhibits higher ATPase activity than Hsp90 from non-cancerous cell lines.
Geldanamycin, a natural product, is an Hsp 90 inhibitor that binds to the ATP binding site of Hsp90 inhibiting ATP hydrolysis but not substrate protein binding. Substrate proteins that reside longer on Hsp90 when ATP hydrolysis is inhibited are ubiquinated, and subsequently degraded. Disrupting the function of the Hsp90 complex has been shown to deplete oncogenic kinases (via ubiquitin-mediated proteasomal degradation) and decrease tumor growth. The Hsp90 complex present in tumor cells exhibits much higher affinity for geldanamycin and for 17-AAG, a geldanamycin derivative, than Hsp90 in non-tumor cells. Thus inhibitors of the Hsp90 complex have the ability to convert this protein from a chaperone that insures correct protein folding of oncogenic proteins to a selective protein degradation tool.
Because of its roles in cell cycle control, cell growth, and oncogenesis the Hsp90 complex is an important target for anti-cancer therapeutics. The ability of certain Hsp90 complex inhibitors to cause this protein complex to selectively target its substrate proteins for degradation makes the Hsp90 complex an especially desirable anti-cancer target. Hsp90 is also a potential drug target for autoimmune and degenerative disease because of its role in modulating the cellular stress response.